Chemical, Biological, and Physical Methods
257
Nucleic Acid (DNA) Probes
A DNA probe consists of the DNA sequence from an organism of interest that can be used to
detect homologous DNA or RNA sequences. In effect, the probe DNA must hybridize with that of the
strain to be sought. Ideally, the probe contains sequences that code for a specific product. The probe
DNA must be labeled in some way in order to be able to assess whether hybridization has occurred.
Radioisotopes are the most widely used labels, and they include
32 P,
3 H,
125 I, and
14 C, with
32 P being
the most widely used. Reporter groups such as alkaline phosphatase, peroxidase, fluorescein, haptens,
digoxigenin, and biotin have been developed and used.
46 When biotin is used, its presence is detected
with avidin-enzyme conjugates, antibiotin, or photobiotin. Chromosomal DNA is often the source of
target nucleic acid, but it contains only one copy per cell in most cases. Multiple targets are provided
by mRNA, rRNA, and plasmid DNA, thus making for a more sensitive detection system. Synthetic
oligonucleotide probes may be constructed of 20–50 bases, and under proper conditions, hybridization
times of 30–60 minutes are possible.
In a typical probe application, DNA fragments of unknown organisms are prepared by the use of
restriction endonucleases. After separating fragment strands by electrophoresis, they are transferred to
cellulose nitrate filters and hybridized to the labeled probe. After gentle washing to remove unreacted
probe DNA, the presence of hybridized product is assessed by autoradiography.
The minimum number of bacterial cells that can be detected with a standard probe is in the 10
6 –10
7
range, although some investigators report the detection of 10
4 cells. When probes are used on foods
where perhaps only 1 cfu/ml of the target organism exists, enrichment procedures must be employed to
allow this cell to attain a level that will provide enough DNA for detection. With an initial cell number
of 10
8 cells, probe results may be obtained in around 10–12 hours when radiolabels are employed.
When enrichments are necessary, the time required for results would be enrichment time plus probe
assay time, generally 44 hours or more. Some examples of probe results for foodborne organisms are
summarized below. For a review, see reference 202.
1. For enterotoxigenic C. perfringens in raw beef, a digoxigenin-labeled probe was used to detect
≤10 cfu/g in 48 hours.
10
2. A colorimetric DNA probe was compared to the Food and Drug Administration (FDA) culture
method for L. monocytogenes in dairy, meat, and seafood samples.
21 Of 660 dairy and seafood
samples examined, 354 were found positive by the FDA and 393 were found positive by the
probe. Of 540 meat samples examined, 261 were found positive by the FDA and 378 by the probe.
Probe results could be obtained in 48 hours, whereas 3–4 days were required for the FDA/USDA
method.
3. For Salmonella spp., a colorimetric probe correctly identified all of 110 serovars and gave no
false positives from 61 nonsalmonellae.
38
4. Radiolabeled probe for salmonellae was tested on 269 poultry carcass and water samples, and
as few as 0.03 cell/ml could be detected when two enrichments were used.
86 The probe could
detect as few as ∼10
4 cells per milliliter and it is AOAC approved.
A probe for S. aureus enterotoxins was constructed from DNA encoding amino acids 207–219 of
SEB and SEC reacted with the genes for SEB and 3 SEC enterotoxins.
150
DNA probes are used in colony hybridization methods where microcolonies of macrocolonies of the
target organism are allowed to develop directly on a membrane following incubation on a suitable agar
medium. A replica plate is produced as a duplicate of the master plate or membrane. Colonies that have
257
Nucleic Acid (DNA) Probes
A DNA probe consists of the DNA sequence from an organism of interest that can be used to
detect homologous DNA or RNA sequences. In effect, the probe DNA must hybridize with that of the
strain to be sought. Ideally, the probe contains sequences that code for a specific product. The probe
DNA must be labeled in some way in order to be able to assess whether hybridization has occurred.
Radioisotopes are the most widely used labels, and they include
32 P,
3 H,
125 I, and
14 C, with
32 P being
the most widely used. Reporter groups such as alkaline phosphatase, peroxidase, fluorescein, haptens,
digoxigenin, and biotin have been developed and used.
46 When biotin is used, its presence is detected
with avidin-enzyme conjugates, antibiotin, or photobiotin. Chromosomal DNA is often the source of
target nucleic acid, but it contains only one copy per cell in most cases. Multiple targets are provided
by mRNA, rRNA, and plasmid DNA, thus making for a more sensitive detection system. Synthetic
oligonucleotide probes may be constructed of 20–50 bases, and under proper conditions, hybridization
times of 30–60 minutes are possible.
In a typical probe application, DNA fragments of unknown organisms are prepared by the use of
restriction endonucleases. After separating fragment strands by electrophoresis, they are transferred to
cellulose nitrate filters and hybridized to the labeled probe. After gentle washing to remove unreacted
probe DNA, the presence of hybridized product is assessed by autoradiography.
The minimum number of bacterial cells that can be detected with a standard probe is in the 10
6 –10
7
range, although some investigators report the detection of 10
4 cells. When probes are used on foods
where perhaps only 1 cfu/ml of the target organism exists, enrichment procedures must be employed to
allow this cell to attain a level that will provide enough DNA for detection. With an initial cell number
of 10
8 cells, probe results may be obtained in around 10–12 hours when radiolabels are employed.
When enrichments are necessary, the time required for results would be enrichment time plus probe
assay time, generally 44 hours or more. Some examples of probe results for foodborne organisms are
summarized below. For a review, see reference 202.
1. For enterotoxigenic C. perfringens in raw beef, a digoxigenin-labeled probe was used to detect
≤10 cfu/g in 48 hours.
10
2. A colorimetric DNA probe was compared to the Food and Drug Administration (FDA) culture
method for L. monocytogenes in dairy, meat, and seafood samples.
21 Of 660 dairy and seafood
samples examined, 354 were found positive by the FDA and 393 were found positive by the
probe. Of 540 meat samples examined, 261 were found positive by the FDA and 378 by the probe.
Probe results could be obtained in 48 hours, whereas 3–4 days were required for the FDA/USDA
method.
3. For Salmonella spp., a colorimetric probe correctly identified all of 110 serovars and gave no
false positives from 61 nonsalmonellae.
38
4. Radiolabeled probe for salmonellae was tested on 269 poultry carcass and water samples, and
as few as 0.03 cell/ml could be detected when two enrichments were used.
86 The probe could
detect as few as ∼10
4 cells per milliliter and it is AOAC approved.
A probe for S. aureus enterotoxins was constructed from DNA encoding amino acids 207–219 of
SEB and SEC reacted with the genes for SEB and 3 SEC enterotoxins.
150
DNA probes are used in colony hybridization methods where microcolonies of macrocolonies of the
target organism are allowed to develop directly on a membrane following incubation on a suitable agar
medium. A replica plate is produced as a duplicate of the master plate or membrane. Colonies that have